OECD/OCDE 405 Adopted:

2 October 2012

© OECD, (2012)

You are free to use this material for personal, non-commercial purposes without seeking prior consent from 

the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written 

permission from the OECD.

OECD GUIDELINE FOR THE TESTING OF CHEMICALS

Acute Eye Irritation/Corrosion

INTRODUCTION 

1. OECD Guidelines for Testing of Chemicals are periodically reviewed to ensure that they reflect the 

best available science. In previous reviews of this Test Guideline, special attention was given to possible 

improvements through the evaluation of all existing information on the test substance in order to avoid 

unnecessary testing in laboratory animals and thereby address animal welfare concerns. This Test

Guideline (adopted in 1981 and updated in 1987, 2002, and 2012) includes the recommendation that prior 

to undertaking the described in vivo test for acute eye irritation/corrosion, a weight-of-the-evidence 

analysis be performed (1) on the existing relevant data. Where insufficient data are available, it is 

recommended that they be developed through application of sequential testing (2) (3). The testing strategy 

includes the performance of validated and accepted in vitro tests and is provided as a Supplement to the 

Guideline. Testing in animals should only be conducted if determined to be necessary after consideration 

of available alternative methods, and use of those determined to be appropriate. At the time of drafting of 

this updated TG 405, there are instances where using this Test Guideline is still necessary or required by 

some regulatory authorities.

2. The latest update mainly focused on the use of analgesics and anesthetics without impacting the basic 

concept and structure of the Test Guideline. ICCVAM and an independent international scientific peer 

review panel reviewed the usefulness and limitations of routinely using topical anesthetics, systemic 

analgesics, and humane endpoints during in vivo ocular irritation safety testing (12). The review concluded 

that the use of topical anesthetics and systemic analgesics could avoid most or all pain and distress without 

affecting the outcome of the test, and recommended that these substances should always be used. This Test 

Guideline takes this review into account. Topical anesthetics, systemic analgesics, and humane endpoints

should be routinely used during acute eye irritation and corrosion in vivo testing. Exceptions to their use 

should be justified. The refinements described in this proposal will substantially reduce or avoid animal 

pain and distress in most testing situations where in vivo ocular safety testing is still necessary.

3. Balanced preemptive pain management should include (i) routine pretreatment with a topical anesthetic 

(e.g., proparacaine or tetracaine) and a systemic analgesic (e.g. buprenorphine), (ii) routine post-treatment 

schedule of systemic analgesia (e.g., buprenorphine and meloxicam), (iii) scheduled observation, 

monitoring, and recording of animals for clinical signs of pain and/or distress, and (iv) scheduled 

observation, monitoring, and recording of the nature, severity, and progression of all eye injuries. Further 

detail is provided in the updated procedures described below. Following test substance administration, no 

additional topical anesthetics or analgesics should be applied in order to avoid interference with the study. 
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Analgesics with anti-inflammatory activity (e.g., meloxicam) should not be applied topically, and doses 

used systemically should not interfere with ocular effects. 

4. Definitions are set out in the Annex to the Guideline. 

INITIAL CONSIDERATIONS 

5. In the interest of both sound science and animal welfare, in vivo testing should not be considered until 

all available data relevant to the potential eye corrosivity/irritation of the substance have been evaluated in 

a weight-of-the-evidence analysis. Such data include evidence from existing studies in humans and/or 

laboratory animals, evidence of eye corrosivity/irritation of one or more structurally related substances or 

mixtures of such substances, data demonstrating high acidity or alkalinity of the substance (4) (5), and 

results from validated and accepted in vitro or ex vivo tests for skin corrosion and eye corrosion/irritation 

(6) (13) (14) (15) (16) (17). The studies may have been conducted prior to, or as a result of, a weight-ofthe-evidence analysis. 

6. For certain substances, such an analysis may indicate the need for in vivo studies of the ocular 

corrosion/irritation potential of the substance. In all such cases, before considering the use of the in vivo 

eye test, preferably a study of the in vitro and/or in vivo skin corrosion effects of the substance should be 

conducted first and evaluated in accordance with the sequential testing strategy in Test Guideline 404 (7).

7. A preferred sequential testing strategy, which includes the performance of validated in vitro or ex vivo 

eye corrosion/irritation tests, is included as a Supplement to this Guideline. It is recommended that this 

testing strategy be followed prior to undertaking in vivo testing. For new substances, it is the recommended 

stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of the 

substance. For existing substances with insufficient data on skin and eye corrosion/irritation, the strategy

can be used to fill missing data gaps. The use of a different testing strategy or procedure, or the decision 

not to use a stepwise testing approach, should be justified.

PRINCIPLE OF THE IN VIVO TEST 

8. Following pretreatment with a systemic analgesic and induction of appropriate topical anesthesia, the 

substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the 

untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of

conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are 

also described to provide a complete evaluation of the effects. The duration of the study should be 

sufficient to evaluate the reversibility or irreversibility of the effects. 

9. Animals showing signs of severe distress and/or pain at any stage of the test or lesions consistent with 

the humane endpoints described in this Test Guideline (see Paragraph 26) should be humanely killed, and 

the substance assessed accordingly. Criteria for making the decision to humanely kill moribund and 

severely suffering animals are the subject of a separate Guidance Document (8). 

PREPARATIONS FOR THE IN VIVO TEST 

Selection of species 

10. The albino rabbit is the preferable laboratory animal and healthy young adult animals are used. A 

rationale for using other strains or species should be provided. 
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Preparation of animals 

11. Both eyes of each experimental animal provisionally selected for testing should be examined within 24 

hours before testing starts. Animals showing eye irritation, ocular defects, or pre-existing corneal injury 

should not be used. 

Housing and feeding conditions 

12. Animals should be individually housed. The temperature of the experimental animal room should be 

20°C (± 3°C) for rabbits. Although the relative humidity should be at least 30% and preferably not exceed 

70%, other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the 

sequence being 12 hours light, 12 hours dark. Excessive light intensity should be avoided. For feeding, 

conventional laboratory diets may be used with an unrestricted supply of drinking water. 

TEST PROCEDURE 

Use of topical anesthetics and systemic analgesics

13. The following procedures are recommended to avoid or minimize pain and distress in ocular safety 

testing procedures. Alternate procedures that have been determined to provide as good or better avoidance 

or relief of pain and distress may be substituted.

• Sixty minutes prior to test substance application (TSA), buprenorphine 0.01 mg/kg is administered by 

subcutaneous injection (SC) to provide a therapeutic level of systemic analgesia. Buprenorphine and 

other similar opiod analgesics administered systemically are not known or expected to alter ocular 

responses (12).

• Five minutes prior to TSA, one or two drops of a topical ocular anesthetic (e.g. 0.5% proparacaine 

hydrochloride or 0.5% tetracaine hydrochloride) are applied to each eye. In order to avoid possible 

interference with the study, a topical anesthetic that does not contain preservatives is recommended. 

The eye of each animal that is not treated with a test article, but which is treated with topical 

anesthetics, serves as a control. If the test substance is anticipated to cause significant pain and distress, 

it should not normally be tested in vivo. However, in case of doubt or where testing is necessary, 

consideration should be given to additional applications of the topical anesthetic at 5-minute intervals 

prior to TSA. Users should be aware that multiple applications of topical anesthetics could potentially 

cause a slight increase in the severity and/or time required for chemically-induced lesions to clear.

• Eight hours after TSA, buprenorphine 0.01 mg/kg SC and meloxicam 0.5 mg/kg SC are administered 

to provide a continued therapeutic level of systemic analgesia. While there are no data to suggest that 

meloxicam has anti-inflammatory effects on the eye when administered SC once daily, meloxicam 

should not be administered until at least 8 hours after TSA in order to avoid any possible interference 

with the study (12).

• After the initial 8-hour post-TSA treatment, buprenorphine 0.01 mg/kg SC should be administered 

every 12 hours, in conjunction with meloxicam 0.5 mg/kg SC every 24 hours, until the ocular lesions 

resolve and no clinical signs of pain and distress are present. Sustained-release preparations of 

analgesics are available that could be considered to decrease the frequency of analgesic dosing.

• “Rescue” analgesia should be given immediately after TSA if pre-emptive analgesia and topical 

anesthesia are inadequate. If an animal shows signs of pain and distress during the study, a “rescue” 

dose of buprenorphine 0.03 mg/kg SC would be given immediately and repeated as often as every 8 

hours, if necessary, instead of 0.01 mg/kg SC every 12 hours. Meloxicam 0.5 mg/kg SC would be 

administered every 24 hours in conjunction with the “rescue” dose of buprenorphine, but not until at 

least 8 hours post-TSA. 
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Application of the test substance 

14. The test substance should be placed in the conjunctival sac of one eye of each animal after gently 

pulling the lower lid away from the eyeball. The lids are then gently held together for about one second in 

order to prevent loss of the material. The other eye, which remains untreated, serves as a control. 

Irrigation 

15. The eyes of the test animals should not be washed for at least 24 hours following instillation of the test 

substance, except for solids (see paragraph 18), and in case of immediate corrosive or irritating effects. At 

24 hours a washout may be used if considered appropriate. 

16. Use of a satellite group of animals to investigate the influence of washing is not recommended unless it 

is scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washing 

should be carefully documented, e.g., time of washing; composition and temperature of wash solution; 

duration, volume, and velocity of application. 

Dose level 

(1) Testing of liquids

17. For testing liquids, a dose of 0.1 mL is used. Pump sprays should not be used for instilling the 

substance directly into the eye. The liquid spray should be expelled and collected in a container prior to 

instilling 0.1 mL into the eye. 

(2) Testing of solids 

18. When testing solids, pastes, and particulate substances, the amount used should have a volume of 0.1 

mL or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume of 

solid material should be measured after gently compacting it, e.g. by tapping the measuring container. If 

the solid test substance has not been removed from the eye of the test animal by physiological mechanisms 

at the first observation time point of 1 hour after treatment, the eye may be rinsed with saline or distilled 

water. 

(3) Testing of aerosols 

19. It is recommended that all pump sprays and aerosols be collected prior to instillation into the eye. The 

one exception is for substances in pressurised aerosol containers, which cannot be collected due to 

vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a 

simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance may 

vary depending on the pressure of the spray and its contents. Care should be taken not to damage the eye 

from the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential for 

“mechanical” damage to the eye from the force of the spray. 

20. An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance is 

sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper. 

The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile 

substances, the dose may be estimated by weighing a receiving container before and after removal of the 

test material. 

Initial test (in vivo eye irritation/corrosion test using one animal) 

21. It is strongly recommended that the in vivo test be performed initially using one animal (see 

Supplement to Guideline 405: A Sequential Testing Strategy for Eye Irritation and Corrosion). 

Observations should allow for determination of severity and reversibility before proceeding to a 

confirmatory test in a second animal. 
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22. If the results of this test indicate the substance to be corrosive or a severe irritant to the eye using the 

procedure described, further testing for ocular irritancy should not be performed. 

Confirmatory test (in vivo eye irritation test with additional animals) 

23. If a corrosive or severe irritant effect is not observed in the initial test, the irritant or negative response 

should be confirmed using up to two additional animals. If an irritant effect is observed in the initial test, it 

is recommended that the confirmatory test be conducted in a sequential manner in one animal at a time, 

rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or

severe irritant effects, the test is not continued. If results from the second animal are sufficient to allow for 

a hazard classification determination, then no further testing should be conducted. 

Observation period 

24. The duration of the observation period should be sufficient to evaluate fully the magnitude and 

reversibility of the effects observed. However, the experiment should be terminated at any time that the 

animal shows signs of severe pain or distress (8). To determine reversibility of effects, the animals should 

be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21 

days, the experiment should be terminated at that time. 

Clinical observations and grading of eye reactions 

25. The eyes should be comprehensively evaluated for the presence or absence of ocular lesions one hour 

post-TSA, followed by at least daily evaluations. Animals should be evaluated several times daily for the 

first 3 days to ensure that termination decisions are made in a timely manner. Test animals should be 

routinely evaluated for the entire duration of the study for clinical signs of pain and/or distress (e.g. 

repeated pawing or rubbing of the eye, excessive blinking, excessive tearing) (9) (10) (11) at least twice 

daily, with a minimum of 6 hours between observations, or more often if necessary. This is necessary to (i) 

adequately assess animals for evidence of pain and distress in order to make informed decisions on the 

need to increase the dosage of analgesics and (ii) assess animals for evidence of established humane 

endpoints in order to make informed decisions on whether it is appropriate to humanely euthanize animals, 

and to ensure that such decisions are made in a timely manner. Fluorescein staining should be routinely 

used and a slit lamp biomicroscope used when considered appropriate (e.g., assessing depth of injury when 

corneal ulceration is present) as an aid in the detection and measurement of ocular damage, and to evaluate 

if established endpoint criteria for humane euthanasia have been met. Digital photographs of observed 

lesions may be collected for reference and to provide a permanent record of the extent of ocular damage. 

Animals should be kept on test no longer than necessary once definitive information has been obtained. 

Animals showing severe pain or distress should be humanely killed without delay, and the substance 

assessed accordingly. 

26. Animals with the following eye lesions post-instillation should be humanely killed (refer to Table 1 for 

a description of lesion grades): corneal perforation or significant corneal ulceration including staphyloma; 

blood in the anterior chamber of the eye; grade 4 corneal opacity; absence of a light reflex (iridial response 

grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjunctivae 

or nictitating membrane; or sloughing. This is because such lesions generally are not reversible. 

Furthermore, it is recommended that the following ocular lesions be used as humane endpoints to terminate 

studies before the end of the scheduled 21-day observation period. These lesions are considered predictive 

of severe irritant or corrosive injuries and injuries that are not expected to fully reverse by the end of the 

21-day observation period: severe depth of injury (e.g., corneal ulceration extending beyond the superficial 

layers of the stroma), limbus destruction >50% (as evidenced by blanching of the conjunctival tissue), and 

severe eye infection (purulent discharge). A combination of: vascularization of the cornea surface (i.e., 

pannus); area of fluorescein staining not diminishing over time based on daily assessment; and/or lack of 

re-epithelialization 5 days after test substance application could also be considered as potentially useful 
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criteria to influence the clinical decision on early study termination. However, these findings individually 

are insufficient to justify early study termination. Once severe ocular effects have been identified, an 

attending or qualified laboratory animal veterinarian or personnel trained to identify the clinical lesions 

should be consulted for a clinical examination to determine if the combination of these effects warrants 

early study termination. The grades of ocular reaction (conjunctivae, cornea and iris) should be obtained 

and recorded at 1, 24, 48, and 72 hours following test substance application (Table 1). Animals that do not 

develop ocular lesions may be terminated not earlier than 3 days post instillation. Animals with ocular 

lesions that are not severe should be observed until the lesions clear, or for 21 days, at which time the study 

is terminated. Observations should be performed and recorded at a minimum of 1 hour, 24 hours, 48 hours, 

72 hours, 7 days, 14 days, and 21 days in order to determine the status of the lesions, and their reversibility 

or irreversibility. More frequent observations should be performed if necessary in order to determine 

whether the test animal should be euthanized out of humane considerations or removed from the study due 

to negative results 

27. The grades of ocular lesions (Table 1) should be recorded at each examination. Any other lesions in the 

eye (e.g. pannus, staining, anterior chamber changes) or adverse systemic effects should also be reported. 

28. Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, 

or other suitable device. After recording the observations at 24 hours, the eyes may be further examined 

with the aid of fluorescein. 

29. The grading of ocular responses is necessarily subjective. To promote harmonisation of grading of 

ocular response and to assist testing laboratories and those involved in making and interpreting the 

observations, the personnel performing the observations need to be adequately trained in the scoring 

system used. 

DATA AND REPORTING 

Evaluation of results 

30. The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, 

and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard 

for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, 

individual scores should be viewed as reference values and are only meaningful when supported by a full 

description and evaluation of all observations. 

Test report 

31. The test report should include the following information: 

Rationale for in vivo testing: weight-of-the-evidence analysis of pre-existing test data, including 

results from sequential testing strategy: 

– description of relevant data available from prior testing; 

– data derived in each step of testing strategy; 

– description of in vitro tests performed, including details of procedures, results obtained with 

test/reference substances; 

– description of in vivo dermal irritation / corrosion study performed, including results obtained; 

– weight-of-the-evidence analysis for performing in vivo study 

Test substance: 
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– identification data (e.g. chemical name and if available CAS number, purity, known impurities, source,

lot number); 

– physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability, reactivity with 

water); 

– in case of a mixture, components should be identified including identification data of the constituent 

substances (e.g. chemical names and if available CAS numbers) and their concentrations;

– dose applied;

Vehicle: 

– identification, concentration (where appropriate), volume used; 

– justification for choice of vehicle. 

Test animals: 

– species/strain used, rationale for using animals other than albino rabbit; 

– age of each animal at start of study; 

– number of animals of each sex in test and control groups (if required); 

– individual animal weights at start and conclusion of test; 

– source, housing conditions, diet, etc.

Anaesthetics and analgesics

– doses and times when topical anaesthetics and systemic analgesics were administered;

– if local anaesthetic is used, identification, purity, type, and potential interaction with test substance. 

Results: 

– description of method used to score irritation at each observation time (e.g., hand slitlamp, 

biomicroscope, fluorescein); 

– tabulation of irritant/corrosive response data for each animal at each observation time up to removal of 

each animal from the test; 

– narrative description of the degree and nature of irritation or corrosion observed; 

– description of any other lesions observed in the eye (e.g., vascularization, pannus formation, adhesions, 

staining); 

– description of non-ocular local and systemic adverse effects, record of clinical signs of pain and 

distress, digital photographs, and histopathological findings, if any. 

Discussion of results. 

Interpretation of the results 

32. Extrapolation of the results of eye irritation studies in laboratory animals to humans is valid only to a 

limited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or 

corrosives. 

33. Care should be taken in the interpretation of data to exclude irritation resulting from secondary 

infection. 
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LITERATURE 

(1) Barratt, M.D., et al. (1995), The Integrated Use of Alternative Approaches for Predicting Toxic 

Hazard, ECVAM Workshop Report 8, ATLA 23, 410 - 429. 

(2) de Silva, O., et al. (1997), Evaluation of Eye Irritation Potential: Statistical Analysis and Tier 

Testing Strategies, Food Chem. Toxicol 35, 159 - 164. 

(3) Worth A.P. and Fentem J.H. (1999), A general approach for evaluating stepwise testing strategies 

ATLA 27, 161-177. 

(4) Young, J.R., et al. (1988), Classification as Corrosive or Irritant to Skin of Preparations Containing

Acidic or Alkaline Substance Without Testing on Animals, Toxicol. In Vitro, 2, 19 - 26. 

(5) Neun, D.J. (1993), Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a 

Single pH, J. Toxicol. Cut. Ocular Toxicol. 12, 227 - 231. 

(6) Fentem, J.H., et al. (1998), The ECVAM international validation study on in vitro tests for skin 

corrosivity. 2. Results and evaluation by the Management Team, Toxicology in Vitro 12, pp.483 –

524. 

(7) OECD (2002), Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testing 

of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264070622-en

(8) OECD (2000), Guidance Document on the Recognition, Assessment and Use of Clinical Signs as 

Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental 

Health and Safety Publications, Series on Testing and Assessment No. 19,

(http://www.oecd.org/ehs/test/monos.htm ). 

(9) Wright EM, Marcella KL, Woodson JF. (1985), Animal pain: evaluation and control, Lab Animal,

May/June:20-36.

(10) National Research Council (NRC) (2008), Recognition and Alleviation of Distress in Laboratory 

Animals, Washington, DC: The National Academies Press.

(11) National Research Council (NRC) (2009), Recognition and Alleviation of Pain in Laboratory 

Animals, Washington, DC: The National Academies Press.

(12) ICCVAM (2010), ICCVAM Test Method Evaluation Report: Recommendations for Routine Use 

of Topical Anesthetics, Systemic Analgesics, and Humane Endpoints to Avoid or Minimize Pain 

and Distress in Ocular Safety Testing, NIH Publication No. 10-7514, Research Triangle Park, 

NC, USA: National Institute of Environmental Health Sciences. 

Available: http://iccvam.niehs.nih.gov/methods/ocutox/OcuAnest-TMER.htm

(13) OECD (2004), Test No. 430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test 

(TER), OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264071124-en

(14) OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines 

for the Testing of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264071148-en

(15) OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines 

for the Testing of Chemicals, Section 4, OECD Publishing.
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doi: 10.1787/9789264071148-en

(16) OECD (2009),Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying 

Ocular Corrosives and Severe Irritants, OECD Guidelines for the Testing of Chemicals, Section 4, 

OECD Publishing.

doi: 10.1787/9789264076303-en

(17) OECD (2009b), Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe 

Irritants, Test Guideline No. 438, OECD Guidelines for the Testing of Chemicals, OECD, Paris

(18) U.S. EPA (2003), Label Review Manual: 3rd Edition, EPA737-B-96-001, Washington, DC: U.S.,

Environmental Protection Agency. 

(19) UN (2011), Globally Harmonized System of Classification and Labelling of Chemicals (GHS),

Fourth revised edition, New York & Geneva: United Nations Publications.

(20) EC (2008), Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 

December 2008 on Classification, Labelling and Packaging of Substances and Mixtures, amending 

and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No. 

1907/2006. Official Journal of the European Union L353, 1-1355.
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TABLE 1: GRADING OF OCULAR LESIONS

Cornea Grade

Opacity: degree of density (readings should be taken from most dense area)* 

No ulceration or opacity .............................................................................................................. 0

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details 

of iris clearly visible ................................................................................................................... 1

Easily discernible translucent area; details of iris slightly obscured ........................................... 2

Nacrous area; no details of iris visible; size of pupil barely discernible ..................................... 3

Opaque cornea; iris not discernible through the opacity ............................................................. 4

Maximum possible: 4

* The area of corneal opacity should be noted 

Iris 

Normal......................................................................................................................................... 0

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; 

or injection; iris reactive to light (a sluggish reaction is considered to be an effect.................... 1

Hemorrhage, gross destruction, or no reaction to light ............................................................... 2

Maximum possible: 2

Conjunctivae 

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris) 

Normal......................................................................................................................................... 0

Some blood vessels hyperaemic (injected) ................................................................................. 1

Diffuse,crimson colour; individual vessels not easily discernible............................................... 2 

Diffuse beefy red ......................................................................................................................... 3

Maximum possible: 3

Chemosis 

Swelling (refers to lids and/or nictating membranes) 

Normal......................................................................................................................................... 0

Some swelling above normal....................................................................................................... 1

Obvious swelling, with partial eversion of lids........................................................................... 2

Swelling, with lids about half closed........................................................................................... 3

Swelling, with lids more than half closed.................................................................................... 4

Maximum possible: 4
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ANNEX 

DEFINITIONS 

1. Acid/alkali reserve: For acidic preparations, this is the amount (g) of sodium hydroxide/100 g of 

preparation required to produce a specified pH. For alkaline preparations, it is the amount (g) of sodium 

hydroxide equivalent to the g sulphuric acid/100 g of preparation required to produce a specified pH 

(Young et al. 1988).

2. Non irritants: Substances that are not classified as EPA Category I, II, or III ocular irritants; or GHS 

eye irritants Category 1, 2, 2A, or 2B; or EU Category 1 or 2 (18) (19) (20).

3. Ocular corrosive: (a) A substance that causes irreversible tissue damage to the eye; (b) Substances that 

are classified as GHS eye irritants Category 1, or EPA Category I ocular irritants, or EU Category 1 (18)

(19) (20).

4. Ocular irritant: (a) A substance that produces a reversible change in the eye; (b) Substances that are 

classified as EPA Category II or III ocular irritants; or GHS eye irritants Category 2, 2A or 2B ; or EU 

Category 2 (18) (19) (20).

5. Ocular severe irritant: (a) A substance that causes tissue damage in the eye that does not resolve within 

21 days of application or causes serious physical decay of vision; (b) Substances that are classified as GHS 

eye irritant Category 1, or EPA Category I ocular irritants, or EU Category 1 (18) (19) (20).

6. Tiered approach: A stepwise testing strategy where all existing information on a test substance is 

reviewed, in a specified order, using a weight-of-evidence process at each tier to determine if sufficient 

information is available for a hazard classification decision, prior to progression to the next tier. If the 

irritancy potential of a test substance can be assigned based on the existing information, no additional 

testing is required. If the irritancy potential of a test substance cannot be assigned based on the existing 

information, a step-wise sequential animal testing procedure is performed until an unequivocal 

classification can be made. 

7. Weight-of-the-evidence (process): The strengths and weaknesses of a collection of information are 

used as the basis for a conclusion that may not be evident from the individual data.
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SUPPLEMENT TO TEST GUIDELINE 405

A Sequential Testing Strategy for Eye Irritation and Corrosion 

GENERAL CONSIDERATIONS 

1. In the interests of sound science and animal welfare, it is important to avoid the unnecessary use 

of animals, and to minimise testing that is likely to produce severe responses in animals. All information 

on a substance relevant to its potential ocular irritation/corrosivity should be evaluated prior to considering 

in vivo testing. Sufficient evidence may already exist to classify a test substance as to its eye irritation or 

corrosion potential without the need to conduct testing in laboratory animals. Therefore, utilizing a weightof-the-evidence analysis and sequential testing strategy will minimise the need for in vivo testing, 

especially if the substance is likely to produce severe reactions.

2. It is recommended that a weight-of-the-evidence analysis be used to evaluate existing 

information pertaining to eye irritation and corrosion of substances and to determine whether additional 

studies, other than in vivo eye studies, should be performed to help characterise such potential. Where 

further studies are needed, it is recommended that the sequential testing strategy be utilised to develop the 

relevant experimental data. For substances which have no testing history, the sequential testing strategy 

should be utilised to develop the data needed to evaluate its eye corrosion/irritation. The initial testing 

strategy described in this Supplement was developed at an OECD workshop (1). It was subsequently 

affirmed and expanded in the Harmonised Integrated Hazard Classification System for Human Health and 

Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals 

Committee and the Working Party on Chemicals, in November 1998 (2), and updated by an OECD expert 

group in 2011.

3. Although this testing strategy is not an integrated part of Test Guideline 405, it expresses the 

recommended approach for the determination of eye irritation/corrosion properties. This approach 

represents both best practice and an ethical benchmark for in vivo testing for eye irritation/corrosion. The 

Guideline provides guidance for the conduct of the in vivo test and summarises the factors that should be 

addressed before considering such a test. The sequential testing strategy provides a weight-of-the-evidence 

approach for the evaluation of existing data on the eye irritation/corrosion properties of substances and a 

tiered approach for the generation of relevant data on substances for which additional studies are needed or 

for which no studies have been performed. The strategy includes the performance first of validated and 

accepted in vitro or ex vivo tests and then of Guideline 404 skin irritation/corrosion studies under specific 

circumstances (3) (4). 

DESCRIPTION OF THE STEPWISE TESTING STRATEGY 

4. Prior to undertaking tests as part of the sequential testing strategy (Figure), all available 

information should be evaluated to determine the need for in vivo eye testing. Although significant 

information might be gained from the evaluation of single parameters (e.g., extreme pH), the totality of 

existing information should be assessed. All relevant data on the effects of the substance in question, and 

its structural analogues, should be evaluated in making a weight-of-the-evidence decision, and a rationale 

for the decision should be presented. Primary emphasis should be placed upon existing human and animal 

data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive 

substances should be avoided whenever possible. The factors considered in the testing strategy include:
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5. Evaluation of existing human and/or animal data and/or in vitro data from validated and 

internationally accepted methods (Step 1). Existing human data, e.g. clinical and occupational studies, and 

case reports, and/or animal test data from ocular studies and/or in vitro data from validated and 

internationally accepted methods for eye irritation/corrosion should be considered first, because they 

provide information directly related to effects on the eyes. Thereafter, available data from human and/or 

animal studies investigating dermal corrosion/irritation, and/or in vitro studies from validated and 

internationally accepted methods for skin corrosion should be evaluated. Substances with known 

corrosivity or severe irritancy to the eye should not be instilled into the eyes of animals, nor should 

substances showing corrosive or severe irritant effects to the skin; such substances should be considered to 

be corrosive and/or irritating to the eyes as well. Substances with sufficient evidence of non-corrosivity 

and non-irritancy from previously performed ocular studies should also not be tested in in vivo eye studies.

6. Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally 

related chemicals should be considered, if available. When sufficient human and/or animal data are 

available on structurally related substances or mixtures of such substances to indicate their eye 

corrrosion/irritancy potential, it can be presumed that the test substance will produce the same responses. 

In those cases, the substance may not need to be tested. Negative data from studies of structurally related 

substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/nonirritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches 

should be used to identify the corrosion and irritation potential for both dermal and ocular effects.

7. Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes 

such as ≤2.0 or ≥11.5 may have strong local effects. If extreme pH is the basis for identifying a substance 

as corrosive or irritant to the eye, then its acid/alkaline reserve (buffering capacity) may also be taken into 

consideration (5)(6)(7). If the buffering capacity suggests that a substance may not be corrosive to the eye

(i.e., substances with extreme pH and low acid/alkaline reserve), then further testing should be undertaken 

to confirm this, preferably by the use of a validated and accepted in vitro or ex vivo test (see paragraph 10).

8. Consideration of other existing information (Step 4). All available information on systemic 

toxicity via the dermal route should be evaluated at this stage. The acute dermal toxicity of the test 

substance should also be considered. If the test substance has been shown to be highly toxic by the dermal 

route, it may not need to be tested in the eye. Although there is not necessarily a relationship between acute 

dermal toxicity and eye irritation/corrosion, it can be assumed that if an agent is highly toxic via the dermal 

route, it will also exhibit high toxicity when instilled into the eye. Such data may also be considered 

between Steps 2 and 3.

9. Assessment of dermal corrosivity of the substance if also required for regulatory purposes (Step 

5). The skin corrosion and severe irritation potential should be evaluated first in accordance with Guideline 

404 (4) and the accompanying Supplement (8), including the use of validated and internationally accepted 

in vitro skin corrosion test methods (9) (10) (11). If the substance is shown to produce corrosion or severe 

skin irritation, it may also be considered to be a corrosive or severely irritant to the eye. Thus, no further 

testing would be required. If the substance is not corrosive or severely irritating to the skin, an in vitro or

ex vivo eye test should be performed.

10. Results from in vitro or ex vivo tests (Step 6). Substances that have demonstrated corrosive or 

severe irritant properties in an in vitro or ex vivo test (12) (13) that has been validated and internationally 

accepted for the assessment specifically of eye corrosivity/irritation, need not be tested in animals. It can 

be presumed that such substances will produce similar severe effects in vivo. If validated and accepted in 

vitro/ex vivo tests are not available, one should bypass Step 6 and proceed directly to Step 7. 
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11. In vivo test in rabbits (Steps 7 and 8): In vivo ocular testing should begin with an initial test using 

one animal. If the results of this test indicate the substance to be a severe irritant or corrosive to the eyes, 

further testing should not be performed. If that test does not reveal any corrosive or severe irritant effects, a 

confirmatory test is conducted with two additional animals. Depending upon the results of the confirmatory 

test, further tests may be needed. [see TG 405]
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LITERATURE

(1) OECD (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on 

Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. 

Held in Solna, Sweden, 22 - 24 January 1996 (http://www.oecd.org/ehs/test/background.htm).

(2) OECD (1998) Harmonized Integrated Hazard Classification System for Human Health and 

Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the 

Chemicals Committee and the Working Party on Chemicals, November 1998 

(http://www.oecd.org/ehs/Class/HCL6.htm).

(3) Worth, A.P. and Fentem J.H. (1999). A General Approach for Evaluating Stepwise Testing 

Strategies. ATLA 27, 161-177.

(4) OECD (2002), Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testing 

of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264070622-en

(5) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or 

Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on 

Animals. Toxicol. In Vitro, 2, 19 - 26.

(6) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., 

Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro 

tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 

12, pp.483 – 524.

(7) Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a 

Single pH. J. Toxicol. Cut. Ocular Toxicol. 12, 227 - 231.

(8) OECD (2001) Supplement to Test Guideline 404: A Sequential Testing Strategy for Skin Irritation 

and Corrosion.

(9) OECD (2004), Test No. 430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test 

(TER), OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264071124-en

(10) OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines 

for the Testing of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264071148-en

(11) OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines 

for the Testing of Chemicals, Section 4, OECD Publishing.

doi: 10.1787/9789264071148-en
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(12) OECD (2009),Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying 

Ocular Corrosives and Severe Irritants, OECD Guidelines for the Testing of Chemicals, Section 4,

OECD Publishing.

doi: 10.1787/9789264076303-en

(13 ) OECD (2009b), Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and 

Severe Irritants, Test Guideline No. 438, OECD Guidelines for the Testing of Chemicals, OECD, Paris
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FIGURE

TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION

Activity Finding Conclusion

1 Existing human and/or animal data, 

and/or in vitro data from validated 

and internationally accepted methods

showing effects on eyes

Existing human and/or animal data 

and/or in vitro data from validated 

and internationally accepted methods

showing corrosive effects on skin

Existing human and/or animal data 

and/or in vitro data from validated 

and internationally accepted methods

showing severe irritant effects on 

skin

Severe damage to eyes

Eye irritant 

Not corrosive/not 

irritating to eyes

Skin corrosive

Severe skin irritant

Apical endpoint; consider corrosive to 

eyes. No testing is needed.

Apical endpoint; consider irritating to 

eyes. No testing is needed.

Apical endpoint; considered noncorrosive and non-irritating to eyes. 

No testing required.

Assume corrosivity to eyes. No testing 

is needed.

Assume irritating to eyes. No testing 

is needed

↓

no information available, or 

available information is not 

conclusive

↓

2 Perform SAR for eye

corrosion/irritation

Consider SAR for skin corrosion 

Predict severe damage to 

eyes

Predict irritation to eyes

Predict skin corrosivity

Assume corrosivity to eyes. No testing 

is needed.

Assume irritating to eyes. No testing 

is needed.

Assume corrosivity to eyes. No testing 

is needed.

↓

No predictions can be made, or 

predictions are not conclusive or 

negative

↓

3 Measure pH (buffering capacity, if 

relevant) 

pH ≤ 2 or ≥ 11.5 (with 

high buffering capacity, 

if relevant)

Assume corrosivity to eyes. No testing 

is needed.

↓

2< pH < 11.5, or pH≤2.0 or ≥11.5 

with low/no buffering capacity, if 
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relevant

↓

4 Consider existing systemic toxicity 

data via the dermal route

Highly toxic at 

concentrations that would 

be tested in the eye.

Substance would be too toxic for 

testing. No testing is needed.

↓

Such information is not available, or 

substance is not highly toxic 

↓

5 Experimentally assess skin corrosion 

potential according to the testing 

strategy in OECD Guideline 404 if 

also required for regulatory purposes

Corrosive or severe 

irritant response

Assume corrosive to eyes. No further 

testing is needed.

↓

Substance is not corrosive or 

severely irritating to skin

↓

6 Perform validated and accepted in 

vitro or ex vivo ocular test(s)

Corrosive or severe 

irritant response 

Irritant response

Non-irritant response

Assume corrosive or severe irritant to 

eyes, provided the test performed can 

be used to identify corrosives/severe 

irritants and the substance is within 

the applicability domain of the test. 

No further testing is needed.

Assume irritant to eyes, provided the 

test(s) performed can be used to 

correctly identify corrosive, severe 

irritants, and irritants, and the 

substance is within the applicability 

domain of the test(s). No further 

testing is needed.

Assume non-irritant to eyes, provided 

the test(s) performed can be used to 

correctly identify non-irritants, 

correctly distinguish these from 

substances that are irritants, severe 

irritants, or ocular corrosives, and the 

substance is within the applicability 

domain of the test. No further testing 

is needed.

↓

Validated and accepted in vitro or ex 

vivo ocular test(s) cannot be used to 

reach a conclusion

↓

7 Perform initial in vivo rabbit eye test

using one animal

Severe damage to eyes Consider corrosive to eyes. No further 

testing is needed.
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↓

No severe damage, or no response

↓

8 Perform confirmatory test using one 

or two additional animals

Corrosive or irritating 

Not corrosive or 

irritating

Consider corrosive or irritating to 

eyes. No further testing is needed

Consider non-irritating and noncorrosive to eyes. No further testing is 

needed.